ECoSCIENCE Estimating N 2 fixation in two species of Alnus in interior Alaska using acetylene reduction

In interior Alaskan boreal forests two species of alder, Alnus tenuifolia and Alnus crispa, represent keystone species in floodplain and upland landscapes, respectively, due to the ability of these plants to form symbiotic associations with the nitrogen-fixing actinomycete, Frankia. It is believed that as much as 70% of the nitrogen (N) accumulated during the 200-y successional development of these forests is derived through atmospheric fixation by these species. Estimates of gross N inputs in these and many other ecosystems have traditionally utilized the acetylene reduction assay (ARA), which requires a conversion factor of the ratio of acetylene to N2 reduced by nitrogenase, the primary enzyme. Despite the fact that small variations in the reduction ratio can substantially influence estimates of N inputs, few studies have investigated how it varies spatially and temporally. The present study sought to 1) determine this conversion factor for both species of alder in situ by calibration of the ARA against a N2 uptake method we developed for field use and 2) determine whether the conversion factor varied with the successional stage in which the alders occurred. Averaged across all plants, the ratio of acetylene to N2 reduced was significantly greater in A. crispa. Significant differences in the value of the conversion factor were observed between early succession and later (mid and late) successional sites for both species. Such differences were also observed among replicate sites within and among stages. However, these stage and site differences may also be due to seasonal effects, which could not be controlled for with our design. Specific acetylene reductase activity (SARA) was only correlated with N2 uptake for early successional sites measured early in the growing season, when N2-fixation rates were lowest and the conversion factor was closest to the theoretical value of 4. A significant negative correlation was found between the conversion factor value and the rate of enzyme activity as determined by the N2 uptake method. Two hypotheses are proposed to explain this result: 1) that it is due to changes in the kinetic properties of nitrogenase at high levels of enzyme activity, resulting in an increased affinity of the enzyme for N2 relative to C2H2, and 2) that the concentration of C2H2 used in our ARA was insufficient to saturate nitrogenase.